1. Field of the Invention
The present invention relates to electrospray ionization (ESI) devices at atmospheric pressure coupled with a mass spectrometer, in particular to a special kind of micro-electrospray with spray flows in the range of 0.1 to 100 microliters per minute.
2. Description of the Related Art
Electrospray ionization devices for use in LC/MS (liquid chromatography/mass spectrometry) can be used to isolate, identify, characterize and quantify a wide range of sample molecules, particularly molecules with high masses, such as peptides and proteins.
Over the past two decades, a number of means and methods of electrospray useful to LC/MS have been developed. Today, LC/MS assays are predominantly run using LC flows of 50 to 5000 microliters per minute feeding the ESI source on the mass spectrometer. For these higher LC flow rates, pneumatically assisted electrospray has become the technique of choice. This technique uses a heated sheath gas sharply blown concentrically around the ESI spray tip to assist in the formation, desolvation and finally evaporation of the charged droplets to get an as pure as possible flow of ions of the analyte molecules. The ions are partly highly-charged. Although the gas greatly helps in the formation of the spray and makes the operation of the electrospray ionization easier and more robust, the excess gas dilutes the sample ions, resulting in lower ion transfer efficiency and loss of sensitivity.
In electrospray ionization, the high electric field first draws a consistent and highly charged jet of the spray solution out of the liquid surface at the tip of the spray capillary. This jet of spray solution decays after a few tenths of a millimeter into numerous (roughly 107 to 108 droplets per second) fine highly charged drops with diameters in the range of 1.0 to 2.0 micrometers. The droplets form a cloud quickly undergoing a space-charge driven lateral expansion. In so doing, the droplets become smaller and smaller by a number of effects: ejection-like evaporation of charged solvent molecules (like hydronium ions) and charged analyte molecules, expelling of smaller highly charged droplets, or splitting of droplets, initiated by charge imbalance. All these processes are accompanied by an evaporation cooling of the droplets which has to be compensated by collision heating within the heated sheath gas. In most cases, the droplets finally completely evaporate, leaving behind charged molecules including the charged analyte molecules.
The process, however, does not always end by complete evaporation. If the droplets are too large in the beginning, or the concentration of heavy molecules in a droplet of the spray fluid is too high, the droplet may not evaporate completely in a distance comparable with the diameter of the ion source. The evaporation may stop because droplets may become too cold for further evaporation. At high concentrations within a droplet, multimers of the molecules may be formed which no longer fall to pieces. Gel-like structures may be formed inside the droplet. Some droplets may even become oversaturated, and a sudden crystallization of molecules occurs, so that a further diminishing of the droplet is no longer possible. All these droplets can be made to pass the entrance of the mass spectrometer without going through by not directing the spray towards this entrance but arranging it off-axis. The inertia of the comparatively heavy droplets lets them fly by.
Most of these ESI sources use this off-axis spray to minimize contamination of the mass spectrometer from tiny droplets which do not completely evaporate in the LC effluent. Though highly charged, the droplets with their high inertia fly past the electrically attracting entrance hole to the mass spectrometer. Some ESI sources utilize special temperature controls and gas flows to further reduce contamination of the mass spectrometer and to increase robustness for LC/MS assays, for instance by the use of a sheath gas around the spray beam and a curtain gas shielding the entrance.
Any LC/ESI-MS assay works best, if the droplets contain a maximum number of one molecule with higher molecular weight only. But this rule is quite often broken because it limits the lowest level of detection.
Although increasingly lower limits of detection can be achieved using larger sample sizes in conjunction with the current high flow LC-ESI/MS systems, sample sizes are becoming more limited as more tests need to be run on a limited amount of a patient's biological fluid, such as blood, urine, sputum, etc. With the increasing need for higher sensitivity in these assays, researchers have explored the use of microESI (˜0.1 to 100 microliters per minute) or nanoESI (˜10 to 1000 nanoliters per minute) to achieve the desired lower limits of detection, but these attempts have at least partially failed to provide the precision and robustness required for quantitative bioanalysis.
For lowest flow LC/MS, nanospray ionization (nanoESI) has become the technique of choice (M. S. Wilm and M. Mann, Int. J. Mass Spectrom. Ion Processes, 136-167, 1994; and M. Mann and M. S. Wilm, U.S. Pat. No. 5,504,329). NanoESI utilizes extremely low liquid flows of 10 to 1000 nanoliters per minute only and a very narrow spray tip outlet placed very close to the entrance of the mass spectrometer, which results in the formation of very small spray droplets with diameters in the range of 200 nanometers only. These tiny droplets can, in the overwhelming number of cases, completely evaporate inside the entrance capillary of the mass spectrometer without the assistance of additional gas flows. Although the ion signal provided by nanoESI in conjunction with mass spectrometry is essentially the same as with conventional ESI, mass spectrometry is a concentration sensitive detection technique which makes nanoESI the best technique for high sensitivity applications. Since no additional gas is used in nanoESI, high ion transfer efficiency can be achieved, but at a cost of ease of use and robustness relative to pneumatically assisted electrospray.
When using nanoESI-MS, the liquid flow rate, solvent composition, spray tip voltage, spray tip design, spray tip integrity and the position of the spray tip outlet relative to the entrance hole of the mass spectrometer are all critical for good spray stability which is needed for a proper ionization by droplet generation and droplet evaporation, and stable ion transfer efficiency. NanoESI spray tips are generally fabricated by pulling and cutting fused silica tubing to make the very small ID/OD tips required for stable spray at nanoliter per minute flow rates, but these tips are difficult to reproduce, fragile to handle and easy to clog. Because of these limitations, nanoESI can be difficult to set up and maintain, making it poorly suited for analyses requiring robust operation. Since nanoESI is generally limited to flow rates below 1 μL/min, samples must be separated using nanoLC which has its own share of problems and limitations. NanoLC requires specialized instrumentation and careful attention to details to insure optimal performance. NanoLC columns (<150 μm ID) have limited sample capacity, require specialized sample injection protocols to load large sample volumes and lack the robustness of larger LC columns. Finally, the low flow rates used in nanoLC/nanoESI-MS typically result in longer sample analysis time, making this technique poorly suited to high throughput applications like biomarker validation and pharmaceutical development.
Several attempts have been made to develop commercially viable microESI sources (sometimes called microspray ionization μSI) in an effort to overcome the limitations imposed by nanoESI, but these microESI sources have not been very well accepted. These microESI sources are basically miniaturized versions of pneumatically assisted ESI and operate with 0.1 to 100 microliters per minute. They offer increased stability and work at higher LC flow rates compared with nanoESI, but the added gas flow results in lower ion transfer efficiency and a loss in sensitivity unacceptable for most researchers. The applicants, therefore, have developed a special microESI/MS electrospray apparatus and method that can overcome the limitations imposed by classical ESI, microESI and nanoESI, without compromising the ion transfer efficiency critical to high sensitivity applications. The apparatus is described in U.S. Pat. No. 8,227,750 B1, and introduced into the market under the trade mark “CaptiveSpray™” The gas flow inside the spray chamber of the CaptiveSpray™ ion source is solely governed by the drawing force of the gas flow through the inlet capillary into the vacuum system of the mass spectrometer; there is no additional gas pumping of any kind. This apparatus and method provide simple, robust operation over a wide dynamic flow range and maintain high ion transfer efficiency independent of the LC flow. The aforementioned patent document (U.S. Pat. No. 8,227,750) is fully incorporated herein by reference.
FIG. 4 shows an illustration adapted from U.S. Pat. No. 8,227,750 from which it is evident that the spray capillary 401 and the transfer capillary 407 that leads directly into the vacuum stage of the mass spectrometer (not shown) are aligned coaxially.
The CaptiveSpray™ ion source has proven to be a great alternative to nanoESI sources for high sensitivity proteomics LC/MS applications where all sample components are of interest. In many LC/MS applications, such as bioanalysis, the components of interest are usually present in low concentrations only and represent only a small fraction of the total sample. To detect the components of lowest concentrations, the solution of the sample is used in a rather high concentration, much higher than those for classic ESI. The high concentration in the spray liquid results in the effect that some droplets, containing many molecules of the main components (sometimes called “matrix” components), do not completely disappear by the usual solvent ion evaporation, droplet splitting and final evaporation. By the evaporation process of the solvent, the droplets may become oversaturated, and a kind of crystallization may occur.
The mass spectrometers used for LC/ESI-MS generally are easily contaminated by particulate matter, such as droplets, diminishing the sensitivity of the mass spectrometer. It has been the experience that even CaptiveSpray™ ion sources lead to contamination of the mass spectrometer if spray liquids with higher analyte concentrations are used.